N,N-dichloro substituted aminocarboxylic acids as microbiocides

ABSTRACT

N-chloro nitrogen-containing carboxylic acids have been found to be useful as microbiocides in topical treatment of the skin and mucous membranes.

This is a division of application Ser. No. 438,712 filed Feb. 1, 1974,now U.S. Pat. No. 3,950,536.

The present invention relates to a novel microbiocidal composition andto the treatment of skin and mucous membranes with a microbiocide.

It has been found according to the invention that microbe infections ofthe skin or mucous membrane, e.g., infections caused by bacteria orfungi, can be effectively treated by applying a microbiocidallyeffective amount of an N-chloro nitrogen-containing carboxylic acid(hereinafter termed N-chlorocarboxylic compound for brevity) of theformulae: ##STR1## wherein R is a divalent saturated aliphatichydrocarbon; R₁ is a monovalent saturated aliphatic hydrocarbon, acyl,or chlorine; n is 1 or 2; p is 1 or 2; R' is a divalent saturatedaliphatic hydrocarbon having from 2 to 4 carbon atoms to complete theheterocyclic ring. In preferred embodiments of the invention, R is adivalent saturated aliphatic hydrocarbon (e.g., alkylene or alkylidene)of 1 to 17 carbon atoms; R₁ is alkyl of 1 to 4 carbon atoms; alkanoyl of2-6 carbon atoms; or chlorine; and R' is ethylene -CH₂ - -CH₂ -,trimethylene -CH₂ CH₂ CH₂ -, or tetramethylene -CH₂ CH₂ CH₂ CH₂ -.Preferred subclasses of N-chlorocarboxylic compounds include, forexample, N-chloroaminoalkanoic acid having 2 to 11 carbon atoms and C₂-C₅ alkane bis(N-chloroamino) C₂ -C₅ alkanedioic acid. Preferredcompounds include, by was of illustration, N,N-dichloro-beta-alanine,N,N-dichloro-11-aminoundecanoic acid,N-chloro-2-pyrrolidone-5-carboxylic acid, andN-chloro-N-acetyl-beta-alanine.

The compounds can be applied as such, in admixture with water, inadmixture with triacetin, in admixture with water and triacetin, increams, ointments or together with other conventional pharmaceuticaladjuvants.

The compounds can also be applied conveniently in compositions of thetype set forth in Evans U.S. Pat. No. 2,618,584, the entire disclosureof the Evans patent being hereby incorporated by reference. Thus, therecan be used from 0.1, and lower, to 5, and higher weight per cent of theN-chlorinated compound with 99.9, and higher, to 95, and lower, weightpercent of a citrate ester of the formula: ##STR2## wherein R₁ is amonobasic acyl group of up to 12 carbon atoms and R₂ is alkyl of up to12 carbon atoms, e.g., acetyl triethyl citrate. There can also be usedesters of glycolic acid, malic acid and tartaric acid, e.g., diacetyldiethyl tartrate, acetyl ethyl malate, acetyl ethyl glycolate. There canalso be used mixtures of these esters with triacetin, e.g., 1 to 99% byweight of either component as solvents for the N-chloroaminocarboxylicacid.

In general, microbiocidal activity has been observed by usingconcentrations as low as 0.1 weight percent, and lower, of theN-chlorocarboxylic compound, based on the weight of the resultingadmixture. Practical considerations such as economics, skin sensitivity,etc., dictate an upper concentration limit of 5 weight percent, andsometimes slightly higher. A concentration range of from about 0.2 toabout 3.0 weight percent is desirable for most cases.

The N-chloro carboxylic compounds can be formed in conventional fashionby reacting a positive halogen source such as a hypochlorite, e.g.,t-butyl hypochlorite, isopropyl hypochlorite, ethyl hyopochlorite,calcium hypochlorite, lithium hypochlorite, sodium hypochlorite,hypochlorous acid, etc.; an N-chloromide, e.g., N-chlorosuccinimide,trichloroisocyanuric acid, etc.; or chlorine gas; with anaminocarboxylic acid or derivative such as glycine, sarcosine,alpha-amino-isobutyric acid, 4-aminobutyric acid, alanine, β-alanine,phenylalanine, norvaline, leucine, isoleucine, 11-amino-undecanoic acid,glutamic acid, valine, glutamine, lysine, alpha-aminobutyric acid,gamma-aminobutyric acid, alpha, epsilon-diaminopimelic acid, ethylenediaminodiacetic acid, 2-pyrrolidone-5-carboxylic acid,N-acetylbeta-alanine, N-caproyl-beta alanine, N-propionyl-beta alanine,N-acetyl glycine, 11-acetamido undecanoic acid, 18-aminostearic acid,18-acetamidostearic acid, N-acetyl phenylalanine, N-ethyl alanine,N-ethyl glycine, 4-aminovaleric acid, caprolactam-7-carboxylic acid andvalerolactam-6-carboxylic acid.

Examples of the N-chloro carboxylic compounds includeN,N-dichloroglycine, N-chlorosarcosine,N,N-dichloroalpha-aminoisobutyric acid, N,N-dichloro-4-amino-butyricacid, N,N-dichloro-DL-alanine, N,N-dichloro-beta-alanine,N,N-dichlorophenylalanine, N,N-dichloronorvaline, N,N-dichloroleucine,N,N-dichloroisoleucine, N,N-dichloro-11-aminoundecanoic acid,N,N-dichloroglutamic acid, N,N-dichlorovaline, N,N-dichloroglutamine,N,N-dichlorotryptophan; N,N-dichlorolysine,N,N-dichloro-alpha-aminobutyric acid, N,N-dichloro-gamma-aminobutyricacid, N,N,N',N'-tetrachloro-alpha,epsilon-diaminopimelic acid,N,N'-dichloro-ethylenediaminodiacetic acid,N-chloro-2-pyrrolidone-5-carboxylic acid,N-chloro-N-acetyl-beta-alanine, N-chloro-N-caproyl-beta alanine,N-chloro-N-propionyl beta alanine, N-chloro-N-acetyl glycine,N-chloro-N-butyryl glycine, N-chloro-11-acetylaminoundecanoic acid,N,N-dichloro-18-aminostearic acid, N-chloro-18-acetylaminostearic acid,N-chloro-N-acetyl phenylalanine, N-chloro-N-ethyl alanine,N-chloro-N-ethyl glycine, N,N-dichloro-4-aminovaleric acid,N-chlorocaprolactam-7-carboxylic acid (2-homopiperidone-7-carboxylicacid) and N-chlorovalerolactam-6-carboxylic acid(2-piperidone-6-carboxylic acid). The preferred compounds are composedof C, H, O and N in addition to the N-chlorine atoms.

Typical examples of preparing the N-chloroaminocarboxylic acids are setforth below.

EXAMPLE 1

7.5 g of glycine is added to 75 ml of methanol and the mixture cooled tobelow 0° C. 26 ml of t-BuOCl is added dropwise, maintaining thetemperature below 0° C. The reaction is followed by the gradualdisappearance of the solid insoluble glycine to form a homogeneoussolution of N,N-dichloroglycine. ##STR3##

The methanol and the resulting t-butanol are stripped off under vacuumand the product obtained as a yellow-white powder with a strong chlorineodor. The yield is 98% and the purity, as determined by titration withNa₂ S₂ O₃ after treatment with KI, is 97%.

EXAMPLE 2

8.7 g Sarcosine is dissolved in 50 ml water and 150 ml of 5% sodiumhypochlorite solution at 5° C. The reaction medium is acidified by HClto pH=1 at low temperature, and 350 ml diethyl ether is added. Theorganic solvent is separated, dried with Na₂ SO₄ stripped off undervacuum. The N-chlorosarcosine is obtained as a white powder in 80% yieldand 90% purity.

The extraction of the N-chloro carboxylic compound from the aqueoussolution may be aided by the addition of NaCl to aid in salting out theN-Cl derivative. A variety of organic solvents can be used to extractthe compounds, including diethylether, chloroform, methylene chloride,carbon tetrachloride and a mixture of these solvents.

EXAMPLE 3

19 g of β-alanine is added to 150 ml methanol. The suspension is stirredand cooled to -25° C. 26 ml of t-BuOCl is added dropwise whilemaintaining the temperature at -°° to 30° C. The gradual disappearanceof most of the insoluble starting material indicates the progression ofthe reaction. The reaction is permitted to warm to room temperature withconstant agitation and the insoluble β-alanine filtered. The methanolsolution of N,N-dichloro-β-alanine is stripped of solvent to yield agreen viscous liquid product in 94% yield and 91% purity.

EXAMPLE 4

5g of L-2-pyrrolidone-5-carboxylic acid is added to 30 ml methanol at10° C. 4 ml of t-butyl hypochlorite is added dropwise while maintainingthe temperature below 25° C. The resulting suspension is stirred for 0.5hour whereupon there is formed a solution. The solution is stripped ofsolvent at room temperature, i.e., 22° C., under approximately 1 mm ofHg. There is obtained N-chloro-L-2-pyrrolidone-5-carboxylic acid in 95%purity and 100% yield.

Examples of other acid precursors reacted are given in Table A.

                                      TABLE A                                     __________________________________________________________________________    REACTANT         PRODUCT      % YIELD                                         __________________________________________________________________________     ##STR4##                                                                                       ##STR5##    80                                               ##STR6##                                                                                       ##STR7##    87                                               ##STR8##                                                                                       ##STR9##    89                                              6-Aminocaproic acid                                                           H.sub.2 N(CH.sub.2).sub.5 CO.sub.2 H                                                           Cl.sub.2 N(CH.sub.2).sub.5 CO.sub.2 H                                                      90                                              Glutamic acid                                                                  ##STR10##                                                                                      ##STR11##   93                                              11-Aminoundecanoic acid                                                       H.sub.2 N(CH.sub.2).sub.10 CO.sub.2 H                                                          Cl.sub.2 N(CH.sub.2).sub.10 CO.sub.2 H                                                     94                                              Ethylenediaminediacetic acid                                                   ##STR12##                                                                                      ##STR13##   94                                              L-2-pyrrolidone-5-carboxylic                                                   acid                                                                          ##STR14##                                                                                      ##STR15##   91                                              __________________________________________________________________________

The compounds used in the present invention did not cause skinirritation in either animal or human tests. They are useful in treatingmicrobe caused diseases of the skin and mucous membranes in mammals suchas humans, mice, rats, cattle, dogs, sheep, cats, rabbits, horses, pigs,etc.

EXAMPLE 5

In the following Example, four Test Solutions were inoculated withviable cells, i.e., Staphylococcus aureus, saliva, Escherichia coli,Streptococcus mutans, Canida albicans, and Aspergillus niger. The fourTest Solutions were as follows:

i. Solution of 1.0 weight percent of N,N-dichlorobeta-alanine in amixture of water and triacetin. The volume of triacetin to water is1:14.

ii. Solution of 0.5 weight percent of N,N-dichlorobeta-alanine in amixture of water and triacetin. The volume of triacetin to water is1:14.

iii. Solution of 0.1 weight percent of N,N-dichlorobeta-alanine in amixture of water and triacetin. The volume of triacetin to water is1:14.

iv. Solution of 0.01 weight percent of N,N-dichlorobeta-alanine in amixture of water and triacetin. The volume of triacetin to water is1:14.

The test organisms and inocula levels used in the experiments areidentified below:

    ______________________________________                                        Test Organisms Viable Cells/Ml of Test Solution                               ______________________________________                                        Staphylococcus aureus                                                                        10 × 10.sup.8                                            T. mentagrophytes                                                                            75 × 10.sup.5                                            S. cerevisiae  14 × 10.sup.7                                            S. faecalis     9 × 10.sup.8                                            Canadian albicans                                                                            40 × 10.sup.6                                            Aspergillus niger                                                                            16 × 10.sup.6                                            ______________________________________                                    

The test method employed was as follows: Nine milliliters of each testsolution was inoculated under ambient conditions, e.g., about 22° C.,with approximately one billion viable cells per milliliter. After 1, 5and 10 minutes, one milliliter aliquots were transferred from the abovesaid inoculated mixtures into 9 ml of neutralizing solution. Theresulting solutions were subsequently serially diluted in 0.1 weightpercent peptone water. Plates were poured with Tryptic Soy Agar for thebacteria and incubated at 37° C. for 48 hours. Yeast plates were pouredwith Mycophil Agar and incubated at 32° C. for 48-72 hours.

The neutralizing solution used to stop the bactericidal (or fungicidal)activity of the test organism is made by admixing 11.9 weight percentTween 80, 1.7 weight percent Azolectin, 3 weight percent Tamol, and 1weight percent peptone in water. The resulting mixture is boiled forabout 15 minutes or until all components are dissolved. Thereafter, itis dispensed in desired volumes such as 9 ml, 99 ml, etc., andautoclaved at 250° F., 15 psig, for 15 minutes. Immediately upon removalfrom the sterilizer, i.e., while still very hot, the bottles should bewell shaken since Tween tends to come out of solution duringautoclaving. The function of this neutralizing solution is to render abactericidal agent inactive by dispersing the molecules that give it itsbactericidal properties. Upon cooling to room temperature, 0.1 gramssodium thiosulfate is added to each 9 milliliters of neutralizer. Thefunction of the sodium thiosulfate is to reduce the active chlorine onthe amino nitrogen atom of the chlorinated amino carboxylic acid.

Peptone is a nitrogen-containing nutrient by Difco Laboratories (DifcoManual of Dehydrated Culture Media and Reagents for Microbiological andClinical Laboratory Procedure, Difco Laboratory Inc., 9th Edition,Detroit, Michigan, page 256 (1971)). Tween 80 is an emulsifier andsolubilizer, polyoxyethylene 20 sorbitan monooleate sold by AtlasChemical Company, Azolectin is lecithin sold by Associated ConcentratesCompany. Tamol is the sodium salt of condensed naphthalene sulfonic soldby Rohm and Haas Corporation.

The controls are described in further detail below. One milliliter ofeach Test Solution was placed in separate bottles containing 9 mlneutralizing solution and were well shaken. One and 0.1 ml aliquots ofthe resulting solutions were placed in sterile petri dishes. Each testorganism was diluted to contain approximately 1,000 cells per ml.One-tenth ml. containing approximately 100 cells were placed directly onthe above said 1 and 0.1 ml. aliquots. After 30 minutes, plates werepoured with respective agars and incubated at appropriate temperatures.Organism controls consisted of placing 0.1 ml. of each cell suspensionin a petri dish and poured with designated agars. After incubation,plates were counted to determine the number of organisms surviving thetest solution plus exposure to neutralizing solution.

The number of viable organisms after exposure to varying concentrationsN,N-dichloro-beta-alanine, i.e., the four Test Solutions, at the end ofeach time period (1 minute, 5 minutes, and 10 minutes) is set forth inTables I through VI below.

Similar tests were carried out with other N-chloroaminocarboxylic acidswithin the invention with the results set forth below. The mixturescontaining the N-chloroamino-carboxylic acids contained water andtriacetin in the same ratio as was used with theN,N-dichlorobeta-alanine, i.e., 1 ml of triacetin and 14 ml of waterplus sufficient of the N-chloroaminocarboxylic acid to give theindicated concentration. The effect on microorganisms of the mixture ofwater and triacetin alone is shown in Table X. It can be seen that thismixture by itself is ineffective as a microbiocide.

The percentages and parts unless otherwise indicated are by weight.

In primary skin irritation in albino rabbits, tests made with 0.05%,N,N-dichloro-beta-alanine-triacetin, 0.1%N,N-dichloro-beta-alanine-triacetin, 0.5%N,N-dichloro-beta-alanine-triacetin, 1%N,N'-dichloro-beta-alanine-triacetin and 3%N,N-dichloro-beta-alanine-triacetin showed no primary skin irritation.No edema or erythema was observed after either 24 or 72 hours in eitherintact or abraded sites. The test method was essentially that of Draizeet al "Methods for the Study of Irritation and Toxicity of SubstancesApplied Topically to the Skin and Mucous Membranes," Journal Pharm. &Exp. Ther.,

                                      TABLE I                                     __________________________________________________________________________                         Activity After Test (Staphylococcus aureus 10                                 × 10.sup.8)                                        __________________________________________________________________________    Test Solution        1 Minute 5 Minutes                                                                              10 Minutes                             __________________________________________________________________________    0.1  Wt. %-N,N-dichloro-β-alanine                                                             < 100    < 100    < 100                                  0.01 Wt. %-N,N-dichloro-β-alanine                                                             17 × 10.sup.4                                                                    57 × 10.sup.4                                                                    39 × 10.sup.3                    __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________                         Activity After Test (C. albicans 40                      __________________________________________________________________________                         × 10.sup.6)                                        Test Solution        1 Minute                                                                             5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    0.1  Wt. %-N,N-dichloro-β-alanine                                                             15 × 10.sup.2                                                                  < 100  < 100                                      0.01 Wt. %-N,N-dichloro-β-alanine                                                             12 × 10.sup.6                                                                  33 × 10.sup.5                                                                  18 × 10.sup.5                        __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________                         Activity After Test (T. mentagrophytes 75 ×                             10.sup.6)                                                __________________________________________________________________________    Test Solution        1 Minute 5 Minutes                                                                              10 Minutes                             __________________________________________________________________________    1.0 Wt. %-N,N-dichloro-β-alanine                                                              < 100    < 100    < 100                                  0.5 Wt. %-N,N-dichloro-β-alanine                                                              < 100    < 100    < 100                                  0.1 Wt. %-N,N-dichloro-β-alanine                                                              < 100    < 100    < 100                                  __________________________________________________________________________

                                      TABLE IV                                    __________________________________________________________________________                        Activity After Test (A. niger 16 × 10.sup.6)        __________________________________________________________________________    Test Solution       1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    1.0 Wt. %-N,N-dichloro-β-alanine                                                             < 100   < 100  < 100                                      0.5 Wt. %-N,N-dichloro-β-alanine                                                             < 100   < 100  < 100                                      0.1 Wt. %-N,N-dichloro-β-alanine                                                             50 × 100.sup.4                                                                  < 100  < 100                                      __________________________________________________________________________

                                      TABLE V                                     __________________________________________________________________________                        Activity After Test (S. cerevisiae 14 ×                                 10.sup.7)                                                 __________________________________________________________________________    Test Solution       1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    1.0 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      0.5 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      0.1 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      __________________________________________________________________________

                                      TABLE VI                                    __________________________________________________________________________                        Activity After Test (S. facecalis 9                       __________________________________________________________________________                        × 10.sup.8)                                         Test Solution       1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    1.0 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      0.5 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      0.1 Wt.%-N,N-dichloro-β-alanine                                                              < 100   < 100  < 100                                      __________________________________________________________________________

                                      TABLE VII                                   __________________________________________________________________________                Staphylococcus aureus 78  × 10.sup.8 (gram +) - Test                    Organisms                                                         Test Compound                                                                             Number of Organisms Recovered After:                              N,N-Dichloroglycine                                                                       1 Minute   5 Minutes  10 Minutes                                  __________________________________________________________________________    20,000 ppm = 2.0 %                                                                        < 10       < 10       < 10                                         5,000 ppm = 0.5 %                                                                        < 10       < 10       < 10                                         1,000 ppm = 0.1 %                                                                        < 10       < 10       < 10                                        __________________________________________________________________________                Streptococcus faecalis 15 × 10.sup.9 (gram +) - Test                    Organisms                                                         Test Compound                                                                             Number of Organisms Recovered After:                              N,N-Dichloroglycine                                                                       1 Minute   5 Minutes  10 Minutes                                  __________________________________________________________________________    20,000 ppm = 2.0 %                                                                        < 10       < 10       < 10                                         5,000 ppm = 0.5 %                                                                        < 10       < 10       < 10                                         1,000 ppm = 0.1 %                                                                        < 10       < 10       < 10                                        __________________________________________________________________________                Trycophyton mentagrophytes 65 × 10.sup.4 (fungus) -                     Test Organisms                                                    Test Compound                                                                             Number of Organisms Recovered After:                              N,N-Dichloroglycine                                                                       1 Minute   5 Minutes  10 Minutes                                  __________________________________________________________________________    20,000 ppm = 2.0 %                                                                        < 10       < 10       < 10                                         5,000 ppm = 0.5 %                                                                        < 10       < 10       < 10                                         1,000 ppm = 0.1 %                                                                        < 10       < 10       < 10                                        __________________________________________________________________________

                                      TABLE VIII                                  __________________________________________________________________________                 S. aureus 8 × 10.sup.8 - Test Organisms                    Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroethylene-                                                         diamino diacetic acid                                                                      1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         INSOLUBLE                                                         2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          TNTC*   TNTC   TNTC                                              __________________________________________________________________________                 P. vulgaris 25 × 10.sup.7 - Test Organisms                 Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroethylene-                                                         diamino diacetic acid                                                                      1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         INSOLUBLE                                                         2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          37 × 10.sup.3                                                                   28 × 10.sup.3                                                                  10                                                __________________________________________________________________________                 C. albicans 49 × 10.sup.6 - Test Organisms                 Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroethylene-                                                         diamino diacetic acid                                                                      1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = INSOLUBLE                                                         2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.20%                                                                          94 × 10.sup.5                                                                   72 × 10.sup.5                                                                  91 × 10.sup.5                               __________________________________________________________________________                 A. pullulans 22 × 10.sup.4 - Test Organisms                Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroethylene-                                                         diamino diacetic acid                                                                      1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         INSOLUBLE                                                         2,000 ppm = 0.2%                                                                          < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          16 × 10.sup.2                                                                   37 × 10.sup.2                                                                  23 × 10.sup.2                               __________________________________________________________________________     *TNTC = Too Numerous To Count                                            

                                      TABLE IX                                    __________________________________________________________________________                 S. aureus 85 × 10.sup.8 - Test Organisms                   Test Compound                                                                              Number of Organisms Recovered After:                             N,N-Dichloroamino-                                                            undecanoic Acid                                                                            1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         410     30     < 10                                              5,000 ppm = 0.5 %                                                                          76 × 10.sup.3                                                                   13 × 10.sup.2                                                                  < 10                                              1,000 ppm = 0.1 %                                                                          44 × 10.sup.5                                                                   320    < 10                                              __________________________________________________________________________                 S. faecalis 16 × 10.sup.9 - Test Organisms                 Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroamino-                                                            undecanoic Acid                                                                            1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         < 10    < 10   < 10                                              5,000 ppm = 0.5 %                                                                          480     < 10   < 10                                              1,000 ppm = 0.1 %                                                                          89 × 10.sup.6                                                                   470    < 10                                              __________________________________________________________________________                 T. mentagraphytes 64 × 10.sup.4 - Test Organisms           Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroamino-                                                            undecanoic Acid                                                                            1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         690     < 10   < 10                                              5,000 ppm = 0.5 %                                                                          24 × 10.sup.3                                                                   < 10   < 10                                              1,000 ppm = 0.1 %                                                                          35 × 10.sup.3                                                                   < 10   < 10                                              __________________________________________________________________________

                                      TABLE X                                     __________________________________________________________________________                 S. aureus - Organism and Inoculum                                Triacetin Control                                                                          Number of Organisms Recovered After:                             1 ml + 14 ml H.sub.2 O                                                                     1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________                 94 × 10.sup.6                                                                   83 × 10.sup.6                                                                  93 × 10.sup.6                               __________________________________________________________________________                 S. faecalis - Organism and Inoculum                              Triacetin Control                                                                          Number of Organisms Recovered After:                             1 ml + 14 ml H.sub.2 O                                                                     1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________                 77 × 10.sup.6                                                                   91 × 10.sup.6                                                                  85 × 10.sup.6                               __________________________________________________________________________                 T. mentagraphytes - Organism and Inoculum                        Triacetin Control                                                                          Number of Organisms Recovered After:                             1 ml + 14 ml H.sub.2 O                                                                     1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________                 42 × 10.sup.4                                                                   46 × 10.sup.4                                                                  48 × 10.sup.4                               __________________________________________________________________________

                                      TABLE XI                                    __________________________________________________________________________                 S. aureus 10 × 10.sup.8 - Test Organisms                   Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloro-4-                                                               amino butyric acid                                                                         1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         < 10    < 10   < 10                                               2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          25 × 10.sup.3                                                                   26 × 10.sup.2                                                                  < 10                                              __________________________________________________________________________                 P. vulgaris 29 × 10.sup.7 - Test Organisms                 Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloro-4-                                                               amino butyric acid                                                                         1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         < 10    < 10   < 10                                               2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          < 10    < 10   < 10                                              __________________________________________________________________________                 Asp. niger 18 × 10.sup.6 - Test Organisms                  Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloro-4-                                                               amino butyric acid                                                                         1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         < 10    < 10   < 10                                               2,000 ppm = 0.2 %                                                                          2 = 10.sup.2                                                                         < 10   < 10                                                200 ppm = 0.02%                                                                          30 × 10.sup.4                                                                   10 × 10.sup.4                                                                  8 × 10                                      __________________________________________________________________________                 C. albicans 26 × 10.sup.6 - Test Organisms                 Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloro-4-                                                               amino butyric acid                                                                         1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    20,000 ppm = 2.0 %                                                                         < 10    < 10   < 10                                               2,000 ppm = 0.2 %                                                                         < 10    < 10   < 10                                                200 ppm = 0.02%                                                                          24 × 10.sup.4                                                                   8 × 10.sup.3                                                                   45 × 10                                     __________________________________________________________________________                 S. aureus - Test Organisms                                       Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroglycine                                                                        1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    1,000 ppm = 0.01%                                                                          < 10    < 10   < 10                                              __________________________________________________________________________                 P. vulgaris - Test Organism                                      Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroglycine                                                                        1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    1,000 ppm = 0.01%                                                                          < 10    < 10   < 10                                              __________________________________________________________________________                 A. niger - Test Organisms                                        Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroglycine                                                                        1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    1,000 ppm = 0.01%                                                                          37 × 10.sup.4                                                                   17 × 10.sup.3                                                                  < 50                                              __________________________________________________________________________                 C. albicans                                                      Test Compound                                                                              Number of Organisms Recovered After:                             N,N-dichloroglycine                                                                        1 Minute                                                                              5 Minutes                                                                            10 Minutes                                        __________________________________________________________________________    1,000 ppm = 0.01%                                                                          47 × 10.sup.4                                                                   < 10   < 10                                              __________________________________________________________________________

                                      TABLE XII                                   __________________________________________________________________________                        S. aureus 12 × 10.sup.8 - Test Organisms            Test Compound       Number of Organisms Recovered After:                      L-N-Chloro-2-Pyrrolidone-                                                     5-carboxylic acid   1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    5,000 ppm = 0.5  wt. %                                                                             10     10     10                                         1,000 ppm = 0.1  wt. %                                                                            180     10     10                                           500 ppm = 0.05 wt. %                                                                            21 × 10.sup.4                                                                   47 × 10.sup.3                                                                  10                                         __________________________________________________________________________                        P. vulgaris - 14 × 10.sup.7 - Test Organisms        Test Compound       Number of Organisms Recovered after:                      L-N-Chloro-2-Pyrrolidone-5-                                                   carboxylic acid     1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    5,000 ppm = 0.5  wt. %                                                                            10      10     10                                         1,000 ppm = 0.1  wt. %                                                                            10      10     10                                           500 ppm = 0.05 wt. %                                                                            10      10     10                                         __________________________________________________________________________                        T. Mentagraphytes 7 × 10.sup.5 - Test                                   Organisms                                                 Test Compound       Number of Organisms Recovered After:                      L-N-Chloro-2-pyrrolidone-                                                     5-Carboxylic Acid   1 Minute                                                                              5 Minutes                                                                            10 Minutes                                 __________________________________________________________________________    5,000 ppm =  0.5  wt. %                                                                           10      10     10                                         1,000 ppm = 0.1  wt. %                                                                            10      10     10                                           500 ppm = 0.05 wt. %                                                                            10      10     10                                         __________________________________________________________________________

Vol. 82, page 377 (1944). At each dosage level there were used sixalbino New Zealand rabbits ranging in weight from 1.8 - 2.4 kg.

The method consisted of applying the test material (0.5 ml) to clippedareas of intact and abraded skin to the extent of approximately 10% ofthe total body surface of the animal. The abrasions were longitudinalepidermal incisions sufficiently deep to penetrate the stratum corneum,but not so deep as to destroy the integrity of the derma. Followingapplication of the test material, the entire trunk of the animal waswrapped in an impervious sheeting. The animals were then immobilized.The sites were individually examined and rated separately for erythemaand edema at 24 and 72 hours. The mean scores for 24 and 72 hourgradings were averaged to determine final irritation indices. As stated,no irritation was noted on a scale ranging from 0 to 4 for eithererythema or edema.

No erythema is 0, very slight erythema is 1 (barely perceptible), on upto severe erythema (beet redness) to slight eschen formation (injuriesin depth) is 4.

Similarly, no edema is 0, very slight edema (barely perceptible) is 1,on up to severe edema (raised more than 1 mm. and extending beyond areaof exposure) is 4.

In in vivo human skin tests using 2%, 1% and 0.5%N,N-dichloro-beta-alanine-triacetin, untreated control, triacetincontrol and skin control the following procedure was used and thefollowing results were obtained:

PROCEDURE

Occlusive patches were made with dermicil hypoallergenic tape andcommercial Saran. Patches measured 2 × 2. One-tenth ml. of each testconcentration was placed on an area of skin marked off at 3/4 × 3/4.

A patch was placed over each test area and remained there for 48 hours.

TEST SAMPLING

After 48 hours each treated area was sampled: 1.5 ml. of 9% Saline wasintroduced into a sterile sampling cup which had been placed well withinthe treated area of skin. The Saline was agitated with a sterile pipettefor 30 seconds after which 1 ml. was withdrawn and placed in 9 ml.peptone water.

Tubes were serially diluted, plated out and poured with Brain Heartinfurion agar.

RESULTS

    ______________________________________                                                Number of Microorganisms per cm.sup.2 Recovered                               From Each Test Area After 48 Hours.                                   2%      0                                                                     1%      0                                                                     0.5%    0                                                                     Untreated Control (Patch placed over untreated skin) 25 × 10.sup.5      Triacetin Control 69 × 10.sup.3                                         Skin Control - To determine no. of cells normally found on                    innoccluded skin - 30                                                         No irritation whatsoever was observed.                                        ______________________________________                                    

The diseases caused by some of the organisms tested are:

    __________________________________________________________________________    C. albicans:                                                                              1)  Thrush (infection of mucous membranes)                                    2)  Cutaneous moniliasis: infection between                                       fingers                                                                   3)  Moniliasis of respiratory tract                               S. aureus:  1)  Food Poisoning                                                            2)  Boils and Carbuncles                                                      3)  Precursors to majority of cases of                                            osteomyelitis and periostitis, otitis                                         media, sinusitis                                                          4)  In animals:                                                                   a. mastitis                                                                   b. staph infection of lambs                                   Strep. faecalis:                                                                          1)  Subacute bacterial endocardites                               T. mentagrophytes:                                                                        1)  Commonest cause of athlete's foot                                         2)  Ringworm of smooth skin                                                   3)  Folliculities of scalp and beard                                          4)  Intertrigeneous infections                                    __________________________________________________________________________

As previously indicated the N-chloro carboxylic compounds employed inthe present invention are preferably applied to skin or mucous membranesin water-triacetin solutions such as those used in the bactericidal andfungicidal tests set forth above. The proportions of materials are notcritical. The N-chloro compound can be present in an amount up to itssolubility. Conveniently they are used at a concentration of 0.01 to 1%.The preferred concentration will vary, of course, with each N-chlorocompound and the particular skin or mucous membrane condition to betreated, i.e., the microorganism involved.

What is claimed is:
 1. A method for treating fungal and bacterialinfections of any area of the skin or mucous membrane comprisingbringing said area into contact with a microbiocidally effective amountof an N-chloro carboxylic acid compound having the formula: ##STR16##wherein R' is a divalent aliphatic hydrocarbon group having 2 to 4carbon atoms.
 2. A method according to claim 1 wherein the infection iscaused by S. aureus, P. vulgaris, T. mentagraphytes, C. albicans, Strep.faecalis, S. cerevisiae or Aspergillus niger.
 3. A method according toclaim 2 wherein R' is ethylene, trimethylene or tetramethylene.
 4. Amethod according to claim 3 wherein the N-chloro carboxylic acidcompound is N-chloro-2-pyrrolidone-5-carboxylic acid.
 5. A methodaccording to claim 1 wherein R¹ is ethylene, trimethylene ortetramethylene.
 6. A method according to claim 1 wherein the compound isN-chloro-2-pyrrolidone-5-carboxylic acid.
 7. A method according to claim6 wherein the N-chloro-carboxylic acid compound is applied to the skin.8. A method according to claim 7 wherein the skin is in the area of thetoes.
 9. A method according to claim 7 wherein the skin is in the areaof the fingers.
 10. A method according to claim 7 wherein the skin is inthe area of the scalp.
 11. A method according to claim 7 wherein theskin is in the area of the face.
 12. A method according to claim 7wherein the N-chloro-carboxylic acid compound isN-chloro-2-pyrrolidone-5-carboxylic acid and the infection is caused byS. aureus, P. vulgaris or T. mentagraphytes.